Cellular Proliferation and Cancer.
The disruption of external or internal regulation of cellular growth can lead to uncontrolled proliferation and in cancer, tumor formation. This loss of control can occur at many levels and, indeed, does occur at multiple levels in most tumors. Further, although tumor cells can no longer control their own proliferation, they still must use the same basic cellular machinery employed by normal cells to drive their growth and replication.
Cyclin Dependent Kinases and Cell Cycle Regulation.
Progression of the normal cell cycle from the G1 to S phase, and from the G2 phase to M phase is dependent on cdks (Sherr, C. J., Science 274:1672-1677 (1996)). Like other kinases, cdks regulate molecular events in the cell by facilitating the transfer of the terminal phosphate of adenosine triphosphate (ATP) to a substrate protein. Isolated cdks require association with a second subunit, called cyclins (Desai et al., Mol. Cell. Biol., 15:345-350 (1995)). Cyclins cause conformational changes at the cdk active site, allowing ATP access and interaction with the substrate protein. The balance between its rates of synthesis and degradation controls the level of each cyclin at any point in the cycle (Elledge, S. J., et al., Biochim. Biophys. Acta, 1377:M61-M70 (1998)). The influences of cyclin/cdk activity on the cell cycle and cellular transformation are summarized in Table 1.
Abnormal Cyclin/cdk Activity in Cancer.
In a normal cell, interlocking pathways respond to the cell's external environment and internal checkpoints monitor conditions within the cell to control the activity of cyclin/cdk complexes. A reasonable hypothesis is that the disruption of normal control of cyclin/cdk activity may result in uncontrolled proliferation. This hypothesis appears to hold in a number of tumor types in which cyclins are expressed at elevated levels (Table 1). Mutations in the genes encoding negative regulators (proteins) of cyclin/cdk activity are also found in tumors (Larsen, C.-J., Prog. Cell Cycle Res., 3:109-124 (1997)); (Kamb, A., Trends in Genetics, 11:136-140 (1995)). Members of the Cip family of cdk inhibitors form a ternary complex with the cyclin/cdk and require binding to cyclinA, cyclinE, or cyclinD (Hall, M., et al., Oncogene, 11:1581-1588 (1995)). In contrast, Ink family members form a binary complex with cdk4 or cdk6 and prevent binding to cyclinD (Parry, D.; et al., EMBO J., 14:503-511 (1995)).
TABLE 1Associations Among Cyclins and CancersCyclinCell Cycle RoleAssociated cdkCancerAS, G2 to Mcdk1, cdk2hepatocellular carcinoma (Wang, J.; etal., Oncogene 8: 1653-1656 (1992))B1/B2G2 to Mcdk1none yet definedD1G1cdk4, cdk6parathyroid adenoma (Motokura, T., etal., Nature, 350: 512-515 (1991))centrocytic B cell lymphoma (Withers,D. A., et al., Mol. Cell. Biol., 11: 4846-4853 (1991))esophageal carcinoma (Jiang, W., et al.,Cancer Res., 52: 2980-2983 (1992))breast cancer (Dickson, C., et al.,Cancer Lett., 90: 43-50 (1995))squamous cell carcinoma (Bartkova, J.,et al., Cancer Res., 55: 949-956 (1995))hepatocellular carcinoma (Nishida, N.,et al., Cancer Res., 54: 3107-3110(1994))D2G1cdk4, cdk6colorectal carcinoma (Leach, F. S., etal., Cancer Res., 53: 1986-1989 (1993))EG1 to Scdk2breast cancer (Keytomarsi, K., et al.,Cancer Res., 54: 380-385 (1994))gastric carcinoma (Akama, Y.; et al.,Jap. J. Cancer Res., 86: 617-621 (1995))colorectal carcinoma (Kitihara, K.; etal., Int. J. Cancer, 62: 25-28 (1995))Inhibitors of Cyclin/cdk Complexes as Potential Anticancer Agents.
Tumors with elevated cyclin/cdk activity, whether from the over expression of cyclins or the loss of an endogenous cdk inhibitor, are prime targets for potential therapies based on small molecule cyclin/cdk inhibitors. In fact, several small molecule inhibitors of cyclin/cdks are reported (Meijer, L., et al., “Progress in Cell Cycle Research,” Plenum Press: New York, 351-363 (1995)) and appear to bind at the ATP site of the kinase. Some information is known about small molecule inhibitors of other kinases, such as PKC (serine kinase) (Murray, K. J. et al., “Ann. Rep. Med. Chem.,” J. Bristol, Ed., Academic Press, Inc.: New York, Chapter 26 (1994)) and tyrosine kinases (Fantl, W. J., et al., Ann. Rev. Biochem., 62:453 (1993); Burke, T. R., Drugs of the Future, 17:119-1131 (1992); Dobrusin, E. M. et al., “Ann. Rep. Med. Chem,” J. Bristol, Ed., Academic Press Inc.: New York, Chapter 18 (1992); Spence, P., Curr. Opin. Ther. Patents, 3:3 (1993)). A number of known inhibitors were obtained from commercial sources or were synthesized by literature procedures.
Purine Compounds as Cyclin/cdk Inhibitors.
There are several reports of 2,6-diamino substituted purine derivatives as cyclin/cdk inhibitors and as inhibitors of cellular proliferation. Among those are reports by U.S. Pat. No. 5,583,137 to Coe, et al., olomoucine (Vesely, J., et al., Eur. J. Biochem., 224:771-786 (1994)), roscovitine (Meijer, L., Eur. J. Biochem., 243:527-536 (1997)), WO 97/16452 to Zimmerman, Imbach, P., et al., Bioorg. Med. Chem. Lett., 9:91-96 (1999), Norman, T. C., et al., J. Amer. Chem. Soc., 118:7430-7431 (1996), Gray, N. S., et al., Tetrahedron Lett., 38:1161-1164 (1997), Gray, N. S., et al., Science, 281:533-538 (1998), WO 98/05335 to Lum, et al., Schow, S. R., et al., Bioorg. Med. Chem. Lett, 7:2697-2702 (1997), U.S. Pat. No. 5,886,702 to Mackman, et al., Nugiel, D. A., et al., J. Org. Chem., 62:201-203 (1997), and Fiorini, M. T. et al., Tetrahedron Lett., 39:1827-1830 (1998). Many of these reported compounds are shown to inhibit cyclin/cdk complexes and have modest cellular proliferation inhibition properties.
The compounds of the present invention are shown to have far superior biological activities as cyclin/cdk complex inhibitors as well as inhibitors of cellular proliferation compared to those previously reported. In fact, the art (e.g., Fiorini, M. T. et al., Tetrahedron Lett., 39:1827-1830 (1998)) teaches away from compounds of this invention, claiming lack of cellular proliferation inhibition.